Excessive activation of the mammalian target of rapamycin (mTOR) and decreased levels of activated STAT3 in diabetic heart lead to higher mortality after acute myocardial infarction. Based on our compelling preliminary data, we hypothesize that mTOR inhibitor, rapamycin, protects against myocardial ischemia-reperfusion (I/R) injury in type 2 diabetes (T2D). Accordingly, in this application, we have proposed novel studies to investigate the mechanisms of cardioprotection with rapamcyin. We will examine the effect of rapamycin treatment (before ischemia or during reperfusion) on myocardial infarct size, function, and cardiomyocytes cell death following I/R in T2D mice. Phosphorylation of STAT3 will be determined in hearts and cardiomyocytes of T2D mice after rapamycin treatment. The essential role of STAT3 in rapamycin-induced protection against I/R injury in hearts and cardiomyocytes will be demonstrated in cardiac-specific STAT3-deficient mice following high-fat diet (HFD)-induced diabetes. Since STAT3 positively regulates miR-20a, we will examine the level of miR-17 and miR-20a, part of miR-17-92 cluster, in diabetic heart and cardiomyocytes with rapamycin treatment. By inducing T2D with the feeding of HFD in cardiac-specific miRNA-17-92 cluster-deficient mice, we will demonstrate cause and effect of miR-17/20a in rapamycin-induced cardioprotection. Finally, we will develop and optimize rapamycin-based therapeutic approach in preventing reperfusion injury in conscious diabetic rabbits. We will test whether rapamycin is effective in reducing infarct size when administered at the time of reperfusion and whether signaling mechanisms similar to db/db mice contribute to cardioprotection in this translational diabetic rabbit model of myocardial infarction. Upon completion of these studies, we expect to gain new insights into the mechanisms involved in cardioprotective effects of rapamycin in the diabetic myocardium. We believe that rapamycin would represent an attractive agent to apply to the clinical setting of acute myocardial infarction in patients. Considering that coronary artery disease is the principal cause of death in diabetic patients, rapamycin therapy may offer a novel therapeutic option for cardioprotection with enormous public health implications.